Sensors and methods of manufacture thereof

ABSTRACT

The invention generally relates to sensors, methods of manufacture thereof, methods of use thereof for sensing analytes, such as small molecules and biomolecules, and methods of immobilization. In certain embodiments, the invention provides a multi-analyte sensor. The multi-analyte sensor includes a plurality of sensing electrodes. Each sensing electrode is functionalized with a different molecule (e.g., biomolecule), at least two of the sensing electrodes are spaced apart prior to and after functionalization by 100 μm or less, and there is no cross-talk between the plurality of sensing electrodes.

RELATED APPLICATION

This application claims the benefit of and priority to U.S. provisionalapplication Ser. No. 61/812,706, filed Apr. 16, 2013, the content ofwhich is incorporated by reference herein its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under RR025761 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention generally relates to sensors, methods of manufacturethereof, methods of use thereof for sensing analytes, such as smallmolecules and biomolecules, and methods of immobilization.

BACKGROUND

Fabrication of electrodes with high spatial resolution and patterningare now mainstream methods in microfabrication and biosensing. Thespatial patterning of different biorecognition molecules (e.g.antibodies, enzymes, aptamers) has not kept pace in practice. By far,the most common methods of biomolecule functionalization of electrodesfor routine fabrication of biosensors for physiology are dip-coating,drop-coating, or spin-coating, none of which can be used tofunctionalize two nearby electrodes (micron scale spacing) with twodifferent biomolecules for multi-analyte sensing.

Top-down approaches such as dip pen lithography or contact printing canbe used, but these are hindered by speed, requirement for highlyspecialized equipment or the need for master templates. Alignmentbetween the electrode and the depositing pen or mask is a primarychallenge that becomes more difficult as spatial features decrease insize. Bottom up approaches typically rely on the chemical selectivity ofthe coupling chemistry to achieve spatially controlled deposition ofspecific biomolecules, limiting the number of unique sensors that can becreated in a small space. In addition, specific coupling chemistries maybe limited to the type of electrode material (e.g. thiol linkages togold) or biomolecule of interest. In order to decorate nearbymicroelectrodes with different recognition biomolecules, a method needsto be developed to selectively control molecular deposition to eachelectrode.

SUMMARY

The invention provides methods of manufacturing sensors in which nearbyelectrodes (e.g., electrodes separated on the micro scale) can beselectively decorated with molecules of interest, such as biomolecules,allowing for the controlled and selective immobilization of themolecules on the nearby electrodes. With methods of the invention,pre-selected electrode sites are decorated with molecules of interestwithout any measurable deposition at a non-addressed nearby electrodesite. Aspects of the invention are accomplished by using the electrodeitself to direct functionalization on specific electrodes byelectrochemical deposition. The methods of the invention involvecontrolling the potential bias on proximate electrodes. A plurality ofelectrode tips are exposed to a medium containing a molecule (e.g., abiomolecule), and only the electrode that is biased during exposure tothe medium is decorated with the molecule. The sensitivity of eachindividual electrode can be tuned by controlling the net charge inputduring deposition, and importantly there is no cross talk between thedifferent electrodes. In that manner, the invention provides sensormanufacturing methods that are simple, fast, tunable, and do not requirehighly specialized equipment. Further, active use of the conductivesubstrate in the immobilization allows the implementation of this methodon various shapes, sizes and 3D orientations of conductive surfaces in ahigh throughput fashion.

In certain aspects, the invention provides methods for functionalizing aplurality of electrodes with different molecules, such as biomolecules.Methods of the invention involve providing first and second electrodes,and introducing the first and second electrodes to a first mediumincluding at least a first molecule and in some embodiments, more thanone type of molecule. The first electrode is electrically biased and thesecond electrode is held at 0 mV or slightly negative, while the firstand second electrodes are both in the first medium, which results inonly the first electrode being functionalized with at least the firstmolecule. The first and second electrodes are then introduced to asecond medium including at least a second molecule. Only the secondelectrode is electrically biased and the first electrode is held at 0 mVor slightly negative, while the first and second electrodes are both inthe second medium, which results in only the second electrode beingfunctionalized with at least the second molecule.

In certain embodiments, after the first electrode is functionalized withthe first molecule (e.g., biomolecule), it is functionalized with asecond molecule (e.g., biomolecule) by electrical biasing in a secondmedium including the second molecule. Various combinations ofelectroactive monomer and molecule (e.g., biomolecule) can be used ineach consecutive layer to achieve specific stacking for the intendedapplication. In certain embodiments, a plurality of molecules areimmobilized per layer. For example two enzymes that work together toconvert an analyte to a product can be placed in a single medium so thatboth enzymes are part of a layer that is formed on a sensing electrode.In another example, a blend of antibodies is immobilized in a singlelayer, which may be useful to detect a ‘fingerprint’ of a disease state.

In certain embodiments, prior to introducing the first and secondelectrodes to the first medium, the method further involves forming anelectrically conductive layer (e.g., a platinum black layer) on thefirst and second electrodes. In certain embodiments, prior tointroducing the first and second electrodes to the first medium, themethod may further involve physically coupling the first and secondelectrodes to each other such that the first and second electrodes arespaced apart by, for example, 100 μm or less prior to and afterfunctionalization. Such physical coupling could be expanded tomulti-wire bundles or chip based electrode arrays for high throughputmulti-molecule immobilization.

The first medium includes an electrically conductive material, such asan electrically conductive polymer. Electrically biasing the firstelectrode may involve applying at least one cycle of cyclical voltage inthe range between 0 mV to 1800 mV to only the first electrode. Theapplication of voltage to the first electrode initiates a polymerizationreaction at only the first electrode that results in production of alayer of the electrically conductive material including the firstmolecule on only the first electrode. The second electrode is held at avoltage between 0 mV and negative 100 mV (−100 mV) to exclude anydepositing layer.

The second medium will also include an electrically conductive material,which may be the same or different from the electrically conductivematerial of the first medium. In certain embodiments, the sameelectrically conductive material, e.g., the same electrically conductivepolymer, is used in the first and second medium. Electrically biasingthe second electrode may involve applying at least one cycle of cyclicalvoltage in the range between 0 mV to 1800 mV to only the secondelectrode. The application of voltage to the second electrode initiatesa polymerization reaction at only the second electrode that results inproduction of a layer of the electrically conductive material includingthe second molecule on only the second electrode. The first electrode isheld at a voltage between 0 mV and −100 mV to exclude any depositinglayer.

Another aspect of the invention provides a multi-analyte sensor. Themulti-analyte sensor includes a control unit and a plurality of sensingelectrodes. Each sensing electrode is functionalized with a differentmolecule, at least two of the sensing electrodes are spaced apart priorto and after functionalization by 100 μm or less, and there is nocross-talk between the plurality of sensing electrodes. The sensor maybe configured to operate in vitro or in vivo.

Numerous different techniques may be used to functionalize theelectrodes with the molecules. In certain embodiments directed togenerating a biosensing electrode and methods of use thereof, eachsensing electrode includes a first layer including an electricallyconductive material, a second layer including a first electricallyconductive biocompatible material, and a third layer including a secondelectrically conductive biocompatible material. In certain embodiments,conductivity of the second electrically conductive biocompatiblematerial is pH dependent. The biomolecule is present in the second andthird layers of each sensing electrode respectively. Numerous differentelectrically conductive materials exist that may be used in the layers,e.g., polymers, semiconductors, metals, etc. In certain embodiments, anelectrically conductive polymer is used in the second and third layers.In certain embodiments, the same electrically conductive polymer is usedin the second and third layers. In other embodiments, a differentelectrically conductive polymer is used in the second and third layers.In an exemplary embodiments, the first layer is platinum black, thefirst electrically conductive biocompatible material ispoly(3,4-ethylenedioxythiophene), and the second electrically conductivebiocompatible material is poly(o-aminophenol). In other embodiments, thesensing electrode is generated using only the second and third layerspreviously described, and excluding the first layer of electricallyconductive material. In further embodiments, the sensing electrodeincludes a layer including a conductive polymer and a first biomolecule,and a second layer including the conductive polymer and a secondbiomolecule different from the first biomolecule. This secondbiomolecule may be used to prevent non-specific interactions with thefirst biomolecule.

Another aspect of the technique allows for controlling the concentrationof molecule coupled to the conductive polymer layer by controlling anumber of voltage cycles. The more cycles applied will result in anincrease in molecule deposition on the sensing electrode. That featureallows a plurality of sensing electrodes to have a gradient of moleculedeposition concentration on the sensor (e.g., a gradient of biomoleculedeposition concentration). That allows, for example, the detection ofrare or small concentrations of a molecule of interest in a sensingenvironment. It can also allow the mapping of concentration gradients ofmolecules of interest in 3D space.

There are numerous different system configurations for the multi-analytesensors of the invention. In one configuration the system includes, acontrol unit, which may optionally include a multi-potentiostat, areference electrode and a counter electrode, and a first sensingelectrode. In other configurations, the multi-analyte sensor comprises asecond sensing electrode.

Another aspect of the invention provides a sensor. The sensor includes acontrol unit and at least one sensing electrode coupled to the controlunit. At least a portion of the sensing electrode includes a first layerincluding an electrically conductive material, a second layer includinga first electrically conductive biocompatible material and a molecule(such as a biomolecule), and a third layer comprising a secondelectrically conductive biocompatible material and the molecule. Incertain embodiments, conductivity of the second electrically conductivebiocompatible material is pH dependent. In certain embodiments, thefirst layer is platinum black, the first electrically conductivebiocompatible material is poly(3,4-ethylenedioxythiophene), and thesecond electrically conductive biocompatible material ispoly(o-aminophenol). The sensor may be configured to operate in vitro orin vivo.

In certain embodiments, the sensor includes a second sensing electrode.The second sensing electrode includes a first layer comprising anelectrically conductive material, a second layer including a firstelectrically conductive biocompatible material and a second molecule(e.g., biomolecule), and a third layer including a second electricallyconductive biocompatible material and the second molecule. In certainembodiments, conductivity of the second electrically conductivebiocompatible material is pH dependent. In certain embodiments, thesensing electrodes are spaced apart by 100 μm or less.

The methods of the invention can be used to couple any molecules (e.g.,biomolecules) known in the art to electrodes, and any molecule known inthe art can be used with any of the sensors of the invention. Exemplarymolecules are biomolecules, and exemplary biomolecules include enzymes,proteins, poly-peptides nucleic acids (e.g., DNA or RNA), antibodies, oraptamers.

In one embodiment the sensor detects electrons produced duringcontrolled reaction to identify and quantify the presence of a moleculeof interest. In another embodiment, the sensing electrode includesfluorescent molecules that are detected optically when there is abinding event between a molecule of interest and the biomoleculeincorporated in a layer of the sensing electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a multi-layer biosensor functionalization scheme. Thethree-layer scheme (Layer 1: Platinum Black, Layer 2: PEDOT+Enzyme,Layer 3: PoAP+Enzyme) allows for controlled functionalization ofelectrode tips with desired enzymes for multi-analyte sensing withenhanced sensitivity and selectivity.

FIG. 2 panels A-B depict an electrode setup & electrodepositionsequence. A custom three-electrode setup (panel A) allows small volumeelectrodeposition of enzyme doped polymer layers to reduce biomoleculewaste. Panel B outlines the electrodeposition sequence for fabricationof a dual analyte stereotrode for simultaneous glucose and lactatesensing. Sequential deposition of three individual sensing layers each(1-3 for glucose sensor on tip 1; 1,4-5 for lactate sensor on tip 2)enables high resolution multi-analyte sensing.

FIG. 3 panels A-F are scanning electro micrographs (obtained using a FEINOVA nanoSEM FESEM under high vacuum with a power setting of 3-5 kV and5-50 kX) of microelectrode at different stages of functionalization showhighly localized deposition of individual sensing layers. (Panel A) Barestereo-electrode with 75 μm tip spacing. (Panel B) Microelectrode tipbefore and (Panel C) after cleaning in H₂SO₄. Morphology of sensor tipafter controlled electrodeposition of Platinum Black (Panel D), PEDOTfilm doped with enzyme (Panel E) and PoAP film doped with enzyme (PanelF). Scale bars in panels A, B-C and D-F are 50 μm, 5 μm and 1 μmrespectively.

FIG. 4 is a graph showing directed electrodeposition of threeconsecutive functionalization layers results in enhanced selectivity forH₂O₂ over Ascorbic Acid (AA). The three layer design allows forindependent control of electroactive surface area enhancement (PtBlack), enzyme doping (PEDOT) and interference rejection (PoAP).

FIGS. 5A-B are graphs showing that glucose and lactate sensors showphysiologically relevant linear calibration ranges. Representativecalibration plots for glucose (5A) and lactate (5B) sensors usingsequential analyte additions at a polarization potential of 0.5V.Sensors show a linear calibration range of 2×10⁻⁵-8×10⁻³ M for Glucoseand 4×10⁻⁶-2.5×10⁻⁴ M for Lactate. Typical response times were 2-5 s.

FIGS. 6A-C are graphs showing that fabricated sensors showed consistentand stable sensitivity to analyte. Sensors fabricated using the samefunctionalization solutions demonstrated similar sensitivities (6A).Sensor response was stable over one week of testing (6B, n=3) with nochange in the linear sensing range (6C).

FIGS. 7A-C are graphs showing that enzyme doping via PEDOTelectrodeposition offers tunable sensitivity. Use of DMSO and elevatedelectrodeposition voltages causes the growth of a PEDOT film withreduced conductivity as shown by the progressively reduced chargeinjection and decreasing area under the cyclic voltammograms (7A).However higher potentials enable tuning of sensor sensitivity withvarying numbers of polymer film growth cycles (7B). Tuning curve islinear with extent of film growth (7C).

FIGS. 8A-B are graphs showing that stereo-electrode sensors show linearsensing ranges similar to single-electrode counterparts and demonstratemulti-analyte capability of functionalization scheme. One microelectrodeeach of the stereo-electrode pair were functionalized with glucoseoxidase (μE1) and lactate oxidase (μE2) for simultaneous glucose andlactate sensing. Shown are concurrently recorded responses of the bothmicroelectrodes of the stereo-electrode pair to additions of glucose(8A) and lactate (8B) at a biasing potential of 0.5 V under rapidstirring.

FIG. 9 is a graph showing that stereo-electrode sensors showed nocrosstalk. Shown are simultaneously recorded responses of the twomicroelectrode sensors to three additions each of lactate and glucose ata biasing potential of 0.5 V. Both sensors respond to changes inconcentration of their respective analytes and show negligible responseto additions of the other analyte. Rapid stirring was used to minimizediffusion related effects and demonstrate the absence of crosstalk.Signal spikes are a result of stirring noise and can be minimized infuture designs by using an additional undoped concentric referenceelectrode.

DETAILED DESCRIPTION

In certain aspects, the invention provides molecule (e.g., biomolecule)deposition methods that can be addressed to specific, pre-selectedelectrode sites without any measurable deposition at a non-addressednearby electrode site. The methods of the invention are simple, fast,tunable, and do not require highly specialized equipment.

FIG. 1 shows an embodiment of an exemplary sensor produced by methods ofthe invention. As shown in FIG. 1, a sensor of the invention generallyincludes at least one sensing electrode coupled to a control unit. Thecontrol unit is not required to be electrically coupled to sensors ofthe invention. For example, in certain embodiments, a depositedbiomolecular layer is used as a biodetection/biorecognition region usingother techniques such as optical (fluorescence/FRET), mass spec, NMR,surface plasmon resonance etc. In those embodiments, the control unit isan entity that is used for detection at some later stage and may or maynot be electrically coupled to the substrate.

A sensor may include more than one sensing electrode, optionallyelectrically coupled to a control unit, generating a multi-analytesensor. The sensing electrodes can have any orientation with respect toeach other. As shown in FIG. 1, the sensing electrodes align with eachother in the same direction, although this is not required. For example,sensing electrodes can be orthogonal to each other. In certainembodiments, the sensor is a chip, such as a microchip, and the sensingelectrodes are incorporated into the microchip. In an exemplaryembodiments, the microchip includes a substrate and the sensingelectrodes are wires having functionalized portions, such asfunctionalized tips. The wires are incorporated into the substrate in amanner in which the functionalized portion of each sensing electrode isable to interact with analytes in a sample.

The sensor may be in any configurable shape, for example, a cube, asphere, or a polygon. The sensing electrodes may be coupled to anon-conducting material, such as plastic, that may be rigid or flexible.Multiple sensors can be coupled together to form a sensor array.

The control unit includes a reference electrode and a potentiostat. Inother embodiments the control unit includes a reference electrode, acounter electrode, and a potentiostat. The potentiostat may be abi-potentiostat or a multi-potentiostat.

FIG. 1 shows first and second electrodes that have been differentlyfunctionalized using methods of the invention. The electrodes are shown75 μm apart, which is only exemplary. Any distance apart of about 100 μmor less can be achieved using methods of the invention. For example, theelectrodes can be 95 μm apart or less, 90 μm apart or less, 85 μm apartor less, 80 μm apart or less, 75 μm apart or less, 70 μm apart or less,65 μm apart or less, 60 μm apart or less, 55 μm apart or less, 50 μmapart or less, 45 μm apart or less, 40 μm apart or less, 35 μm apart orless, 30 μm apart or less, 25 μm apart or less, 20 μm apart or less,etc. As explained in more detail below, the function of the electrodeitself directs functionalization on specific electrodes byelectrochemical deposition of a molecule (e.g., a biomolecule) within amedium including an electroactive biocompatible material (i.e.,electrically conductive material). FIG. 1 and aspects of the applicationdiscuss a specific embodiment of the invention, referring to specificbiomolecules, e.g., enzymes, and specific electroactive biocompatiblematerial, e.g., electroactive biocompatible polymers. The skilledartisan will recognize that FIG. 1 and certain aspects of theapplication herein are exemplary, and not limiting of the scope of theinvention. For example, numerous different electroactive biocompatiblematerials can be used in the methods and sensors described herein, andthe invention is not limited to those materials specifically exemplifiedbelow. Similarly, the methods of the invention can be used to couple anymolecules (inclusive of molecules that are not biomolecules) known inthe art to electrodes, and any molecule known in the art can be usedwith any of the sensors of the invention. Particularly, the invention isnot limited to those biomolecules specifically exemplified below.

Exemplary electroactive biocompatible materials for use with methods andsensors of the invention generally include metals, semi-conductors andpolymers. Exemplary electroactive biocompatible materials arepoly-thiophenes, poly-acetylenes, poly-pyrroles, poly-anilines,poly-indoles, poly-phenylenediamines, poly-phenols, and variousderivatized versions of these polymeric classes.

Exemplary molecules are biomolecules and non-biomolecules. In certainembodiments, the molecule is a biomolecule. Exemplary biomolecules foruse with methods and sensors of the invention generally include enzymes,proteins, poly-peptides, nucleic acids (e.g., DNA and RNA), nucleotides,antibodies, aptamers, small organic molecules, lipids, mono- orpoly-saccharides, glycolipids, sterols, glycerolipids, vitamins,hormones, neurotransmitters, or metabolites. Other biomolecules areknown in the art and are described, for example in Mishra(“Biomolecules”, Discovery Publishing House, Jan. 1, 2003), the contentof which is incorporated by reference herein in its entirety.

Referring back to FIG. 1, which illustrates a specific embodiment of theinvention, the function of the electrode itself directsfunctionalization on specific electrodes by electrochemical depositionof protein within a polymer matrix of two electroactive polymers, namelypoly(3,4-ethylenedioxythiophene) [PEDOT] and poly(o-aminophenol) [PoAP].PEDOT was chosen as its formation from EDOT monomer can be controlled byelectrode potential. PEDOT is also compatible with proteins, and hasbeen recently used in the fabrication of biosensors with improvedconductivity and good thermal and chemical stability. PoAP, the secondpolymer layer used in this functionalization scheme, affords improvedselectivity to the analyte of interest owing to its unique pH dependentconductivity. While several recent studies have shown that electroactivepolymers can be used to enhance sensitivity, selectivity and linearrange of enzyme-based biosensors, most deposition schemes reported todate rely on passive enzyme adsorption at the electrode surface andtherefore lack tunability. Further, these schemes are typicallyimplemented on relatively large electroactive surfaces and theminiaturization and multiplexing of such sensors for multi-analytedetection is still fairly unexplored.

Methods of the invention demonstrate that by proper selection of monomermaterials and deposition parameters, the in situ growth of polymermaterials can be coupled with electro-aided enzyme adsorption to achieveenzyme-based amperometric sensors with tunable sensitivity andselectivity. Further, the same functionalization scheme can be extendedto achieve simple spatially addressable doping of multiple enzymes ofinterest by controlling the potential on proximate electrodes to 0 mV orslightly negative. A fused stereo-microelectrode was designed in whichtwo 1-2 μm diameter microelectrode tips are separated by about 75 μm,which is an only exemplary distance as discussed above (FIG. 1 and FIG.3 panel A).

Both microelectrode tips were exposed to two different PEDOT/enzymesolutions, but only the electrode that is biased during exposure toglucose oxidase became a glucose sensor and only the electrode that wasbiased during exposure to lactate oxidase became a lactate sensor. Thesensitivity of each individual sensor can be tuned by controlling thenet charge input during deposition, and most importantly there was nocross talk between the glucose and lactate detection of the two nearbysensors. This system demonstrates the ability to address specificelectrode sites simply by controlling potential bias thus creating aneasy and tunable method for creating multi-analyte micro-sensors withinsmall sensing areas. In addition, the method is generic to both thesensing element material and the recognition biomolecule and can therebyenable novel patterns of high-density, multi-analyte biosensor arrays.This is the first demonstration of the use of electroactive polymernanocomposites for miniaturized, multiplexed, tunable biomolecule-based(e.g., enzyme-based) biosensors.

Single micro-wire electrodes were chosen as sensor substrates based onprior experience in the use of these materials for the development ofmicro-scale amperometric biosensors. FESEMs of the commercial single andstereo microelectrodes displayed irregular tip profiles (FIG. 3, panelB) that were successfully cleaned and exposed (FIG. 3 panel C) usingseveral potential cycles in sulfuric acid. Electrodeposition of platinumblack onto the exposed tip using a pulsed current scheme resulted in anintricate cauliflower like morphology (FIG. 3, panel D). SubsequentPEDOT and PoAP electrodeposition via potential cycling producedadditional thin film like coatings that were highly localized on theinitial growth of nanostructure platinum (FIG. 3, panels E-F).

The use of three distinct functionalization layers allowed independentcontrol of three key sensor metrics namely, electroactive surface area,transducer doping (discussed below) and interference rejection. FIG. 4outlines the changes in H₂O₂ sensitivity and selectivity of themicrowire after each functionalization step. Electrode cleaning insulfuric acid and electrodeposition of platinum black onto the exposedtip led to over a one-fold increase in electroactive surface area,evidenced by increased H₂O₂ sensitivity and enhanced selectivity overascorbic acid. The second, PEDOT based enzyme entrapment layer showedimproved exclusion of both H₂O₂ and AA, but reduced H₂O₂ sensitivity.The use of a final, enzyme doped PoAP layer served to restoreselectivity owing to the unique permselective properties of this polymerat neutral pH. Together, the two electrodeposited polymer layers allowcontrolled transducer doping and reduced interference from anionicspecies such as ascorbic acid.

The individual parameters for each functionalization layer were firstoptimized for the detection of glucose using the model enzymatictransducer glucose oxidase. Square wave chronopotentiometry was used forplatinum black deposition as it allowed easy control of total injectedcharge. The potential at the electrode was monitored during depositionto ensure repeatability of the final electroactive surface area(conductivity) of the modified tips. The two electroactive polymerlayers were deposited one after another using multiple rounds ofpotential cycling in aqueous enzyme doped monomer solutions. Typicalaqueous electrodeposition strategies for EDOT use negatively chargedpolymers such as PSS to improve monomer solubility and lower depositionpotentials (0 to 1.2V) that rely on passive adsorption for enzymeloading into the growing polymer film. The polar aprotic solventdimethyl sulfoxide (DMSO, 5% v/v) was used in this scheme to obtain astable dispersion of EDOT monomer in aqueous enzyme solution without theuse of additional negatively charged polymers such as PSS. The omissionof PSS prevents competition between PSS and the negatively chargedenzymatic transducer for incorporation into the growing polymer film.Further, the potential range for deposition was raised compared topreviously reported methods, thereby enabling electrochemically aidedadsorption of enzyme, permitting active transducer doping and tunablesensitivity to the analyte of interest. The final permselective PoAPlayer was grown using previously described methods but at higher enzymedoping. The increased enzyme concentrations used in both polymer growthsolutions were found to be necessary to achieve enhanced sensitivity inthe micro-scale form factor.

The resulting glucose sensors were validated via calibration againstknown step additions of glucose at a detection potential of 0.5V. FIG.5A shows the calibration plot for a representative glucose sensor with alinear calibration range of 2×10−5-8×10−3 M. Typical response times werein the range of 2-5 seconds.

Another advantage of the scheme is the ability to produce consistentsensors with similar sensitivities when using the same functionalizationsolutions and electrochemical deposition parameters for the three layer.FIG. 6A shows the responses of two individually functionalized singlewire micro-electrodes to the same concentration increments of glucose.Inter-sensor variability <5% can be easily achieved by monitoringelectrode conductivity during Pt Black electrodeposition and totalcharge injected in each polymer deposition step of the scheme. Further,the stability of resulting sensors was tested by monitoring sensitivityof sensors over a period of one week (FIG. 6B). Sensor responsedecreased by <20% over one week of testing (n=3) when stored at 4° C.Further, the linearity and operating range of the sensors was maintainedover the entire testing period (FIG. 6C).

Finally, the functionalization steps and parameters optimized forglucose sensors were applied to fabricate lactate sensors by simplyreplacing the enzyme in the scheme with lactate oxidase. The resultingsensors showed linear calibration for lactate in the range4×10⁻⁶-2.5×10⁻⁴ M with similar response times at a detection potentialof 0.5V (FIG. 5B).

An important requirement for multi-analyte sensing whether in multi-wireformat or on chip is that the responses from individual analyte sensorsites (in this case current) are all within the measurable range of thepotenstiostat or onboard circuitry. The ability to tune the sensitivity(current response of the sensor per unit change in analyte) of eachspecific site and do so in a repeatable fashion can help avoid complexmeasuring circuitry and maximize the signal to noise ratio of the systemby matching the linear sensing range to the current measurement range.Most electroactive polymer based functionalization schemes reported todate rely on passive adsorption of the enzyme onto the electrode forincorporation into the growing polymer film. The only way to controlenzyme doping in such schemes is by increasing the concentration in thefunctionalization solution. Moreover, as most of the enzyme is typicallyincorporated in the first deposition cycle, the upper limit forsensitivity is defined by the size of the electrode surface. This limitsthe scalability of such schemes and makes it difficult to achievetunable sensitivity in microscale form factors.

Methods of the invention provide for tunable enzyme doping in thisscheme because of several important design decisions pertaining to theelectrodeposition of the PEDOT transducer doping layer as discussedearlier. Selection of appropriate electrolyte pH, elimination ofcompeting negative counter ions such as PSS and use of higherelectrodeposition potentials allowed for active doping of negativelycharged enzymatic transducer. Tunable sensitivity to the analyte ofinterest is easily achieved by varying the number of cycles of EDOT filmgrowth. FIG. 7B. shows a 5 fold increase in sensitivity between the 1stand 20th cycle of electrodeposition for a fixed concentration of enzyme.Further increases in sensitivity are limited by the decrease in theconductivity of the film, and progressive reduction in charge injectedper deposition cycle (FIG. 7A) with increasing cycle number. Higherelectro-deposition potentials have been previously shown to producefilms with reduced conductivity because of the oxidation of theresulting polymer at these potentials.

Plotting the tuning curve for the same sensor shows that analytesensitivity is linearly correlated to the net charge injected (anindicator of the polymer film growth; FIG. 7C). This result is asignificant improvement over existing methods as charge injection iseasily monitored during deposition and provides a simple metric toprecisely control the level of enzyme doping and hence sensitivity ofthe resulting sensor.

As discussed above, the use of electrodeposition allows directedmodification of specific sensing surfaces and can be used formultiplexed or multi-analyte sensing. The deposition methods for thisdesign were expressly selected to enable site-specific functionalizationof multiple enzymes in closely spaced electrodes.

Once the individual parameters for glucose and lactate sensorfunctionalization were optimized using single microwire electrodes theywere used to demonstrate applicability of the scheme for high resolutionmulti-analyte sensing using commercial stereoelectrodes. Both electrodeswere immersed simultaneously in functionalization solutions for bothanalytes. Tunable sensitivity for glucose or lactate was achieved at theelectrode of interest by appropriate selection of enzyme concentrationand number of deposition cycles. Use of a potential of 0V to −100 mVprevented non-specific functionalization and fouling at the oppositeelectrode.

The resulting dual-analyte stereo-electrode was validated inbi-potentiostat mode with both wires simultaneously biased at 0.5Vagainst a common reference electrode. The current responses of bothelectrodes (μE1: glucose sensor, μE2: lactate sensor) were recordedduring standard glucose (FIG. 8A) and lactate (FIG. 8B) calibrationexperiments. Sensor linear ranges and response times showed agreementwith those of single wire sensors, showing that the additional two stepsof functionalization and the use of a potential between 0 mV and −100 mVwas not detrimental to sensor response.

Finally, the responses of the same dual sensor were recorded for threestep additions each of lactate and glucose as shown in FIG. 9. Bothsensors showed strong selectivity for their respective analytes and noresponse to concentration changes of the opposite analyte. Takentogether, these two experiments demonstrate the spatial control affordedby this scheme and demonstrate the applicability of the same tomulti-analyte sensing.

The simple three-layer functionalization scheme described here allowsfor spatially directed functionalization of sensing surfaces forenzyme-based amperometric sensing. Each functionalization layer is shownto serve an important role in defining the characteristics of the finalsensor. The initial platinum black layer greatly enhances the effectivesurface area of the electrode surface while maintaining a small sensingfootprint. The second electrodeposited PEDOT layer is shown to becapable of active transducer doping, allowing the sensitivity to beeasily tuned with no change in linear sensing range. This method is animprovement over previously reported sensing schemes based onelectroactive polymer film growth that rely primarily on passiveadsorption for transducer doping and are hence not easily tunable. Thefinal electrodeposited PoAP layer provides a fold increase inselectivity for H₂O₂ over common interferents such as ascorbic acid. Thecombination of these three functionalization layers affords consistent,spatially controlled doping of transducers for enzyme-based amperometricsensors for high-resolution single/multi-analyte physiological sensing.

Multi-analyte capability of this design is demonstrated using customstereo-electrodes that are functionalized with enzymes for simultaneousglucose and lactate sensing. Both sensing electrodes are addressedsimultaneously and show physiologically relevant linear sensing rangesfor their analytes, negligible crosstalk, fast response times anddecreased interference. These results validate the applicability of thefunctionalization scheme for multi-analyte sensing and pave the way forsimultaneous real-time sensing of multiple analytes in variousphysiological settings.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES

A three-layer functionalization scheme was developed that employselectroactive polymer nanocomposites to achieve tunable, site-directedfunctionalization for enzyme-based biosensors. Nanostructured platinum[Pt Black], enzyme-doped conductive polymerpoly(3,4-ethylenedioxythiophene) [PEDOT] and non-conductive polymerpoly(o-aminophenol) [PoAP] were electrodeposited as consecutive layersto achieve increased electroactive surface area, fine control overlocation and quantity of enzyme, and reduced interference, respectively.Optimization of deposition parameters allowed unprecedented control overenzyme doping and consequently analyte sensitivity of resultingbiosensors. The design was first validated by controlledfunctionalization of a 1-3 μm tip size microelectrodes for glucose andlactate sensing. Resulting sensors showed tunable sensitivity, reducedinterference, physiologically relevant linear sensing ranges, shortresponse times and were stable over the period of testing. Finally,multi-analyte capability of the scheme was demonstrated via simultaneousglucose and lactate sensing using custom stereo-electrodes with 1-3 μmsensing areas and 75 μm tip separation. Use of an exclusively electrodedriven, electrodeposition based functionalization scheme enabled 5×sensitivity tuning, spatially controlled transducer doping unattainablewith conventional dip, drop or spin coating based methods, and allowedthe extension of the scheme to multi-analyte sensing usingmulti-electrode systems and lab-on-chip platforms. The following areexemplary embodiments of the invention.

Example 1: Reagents

Phosphate buffered saline (PBS 0.01 M pH 7.4), chloroplatinic acidsolution (8% wt/wt), lead (II) acetate trihydrate (99%),3,4-Ethylenedioxythiophene (EDOT, 97%, stored at 4° C.), H2O2 (30% (w/w)in H2O, stored at 4° C.), D-glucose (99.5%, BioXtra), dimethyl sulfoxide(99.7%, HPLC grade), sodium acetate buffer solution (3M, pH 5.2), sodiumascorbate and sulfuric acid (98%) were purchased from Sigma-Aldrich (St.Louis, Mo.). Glucose oxidase (Aspergillus niger, lyophilized powder, 220units/mg, stored at 20° C.) and Lactate oxidase (Aerococcus viridianslyophilized powder, 20 units/mg, stored at 20° C.) were obtained fromSekisui Diagnostics (Kent, UK). 2-aminophenol (o-AP, 99%) and sodiumlactate (60% w/w) were purchased from Alfa Aesar (Ward Hill, Mass.). Allbuffers and functionalization solutions, if not specified, were preparedin deionized water (DI) of resistivity 18.2 MΩ cm (Ultrapure water).

Example 2: Apparatus and Electrodes

Platinum/Iridium (Pt/Ir) microelectrodes (PI20033.0A10, MicroprobesInc., Gaithersburg, Md.) with 256 μm diameter shaft, 3 μm parylene Cinsulation, 1-3 μm exposed tip diameter and, 3.0 MΩ impedance were usedfor the design and characterization of the three-layer functionalizationscheme. PEDOT doping tests were conducted using 1.6 mm diameter Pt diskelectrodes (MF-2013, BASi Inc., West Lafayette, Ind.). Custom fabricatedPt/Ir stereotrodes (Microprobes Inc., Gaithersburg, Md.) with 75 μmdiameter shaft, 3 μm parylene C insulation, 1-3 μm exposed tip diameter,75 μm tip separation and, 3.0 MΩ impedance were used to demonstratespatial control and multi-analyte capability of the functionalizationscheme.

All functionalization steps and sensor characterization experimentsunless specified, were performed in a standard three-electrode setupusing a saturated silver/silver chloride reference electrode (BASi Inc.,West Lafayette, Ind.) and a bare platinum wire counter electrode (0.5 mmdia, Alfa Aesar, Ward Hill Mass.). A custom functionalization setup wasused to minimize functionalization solution volumes to 200 μL andoptimize reagent utilization (FIG. 2A).

Example 3: Electrode Pretreatment, Cleaning and Storage

All microwire electrode tips were washed by sonication in Ultrapurewater and dried in a stream of nitrogen gas before modification. Theelectrodes were then immersed in a 1M sulfuric acid solution and thepotential cycled between 0.2 and 1.2 V vs. Ag/AgCl/KCl (sat'd) at 0.1Vs−1 until a steady-state cyclic voltammogram was obtained. Prior to thiselectrochemical pretreatment, the 1.6 mm disk electrodes used tocharacterize enzyme doping were first polished with alumina slurries (1μm/0.3 μm/0.05 μparticles) and then cleaned in Piranha solution toremove organic contaminants between differing functionalization cycles.Following this, all electrodes were again rinsed with liberal amounts ofwater before further processing. Fabricated sensors were rinsed in PBSand stored dry at 4° C. when not in use. Sensors were also rinsedbriefly in PBS before and after all characterization experiments.

Example 4: Platinum Black Deposition

Prior to electrochemical enzyme immobilization, an initial layer ofamorphous platinum black (Pt Black) was grown onto the exposed tip ofmicrowire electrodes to increase effective surface area whilemaintaining small sensing footprint. Electrodes were subjected to pulsedcurrent injection (square wave, 10 ms ON, 500 ms OFF) in a standardthree-electrode setup while immersed in a platinizing solution composedof 17.5 mM Hexachloroplatinic acid and 0.03 mM Lead Acetate. An initialcurrent train of amplitude 10 μA (25 cycles) was used to prime theelectrode and initiate Pt Black deposition followed by a second train ofamplitude 30 μA (100 cycles). The electrodes were then rinsed inultrapure water and dried in a convection oven at 80° C. for 20 minutes.

Example 5: Enzyme Immobilization

Enzyme immobilization on electrode surfaces was achieved throughelectrochemical growth of PEDOT films by cyclic voltammetry (potentialrange 0.8V/1.4V vs. Ag/AgCl/KCl (sat'd) at 50 mV s−1, 1-20 cycles) usinga 5 mM EDOT solution containing 2500 U ml-1 of either glucose or lactateoxidase in Ultrapure water. EDOT was first solubilized in DMSO to make a50 mM stock solution, then diluted to 10 mM using incremental additionsof water and finally vortexed for 20 minutes with an equal volume of theenzyme stock solution (5000 U ml-1) to achieve desired monomer andenzyme concentrations. All electrodes were rinsed in ultrapure water andair-dried prior to further processing.

Example 6: Permselective Poly(o-Aminophenol) Layer

Permselectivity for interferents such as ascorbic acid was achievedusing a final functionalization layer of poly(o-aminophenol) (PoAP)doped again with the enzyme of interest. PoAP films wereelectrochemically deposited via cyclic voltammetry (potential range0V/0.9 V vs. Ag/AgCl/KCl (sat'd) at 50 mV s−1, 15 cycles) using a 5 mMo-AP solution containing 2500 U ml-1 enzyme in 0.05M acetate buffer (pH5.6). The monomer o-AP was first solubilized in 0.1M acetate buffer viasonication to make a 10 mM stock solution that was then vortexed for 20minutes with an equal volume of the enzyme stock solution (5000 U ml-1)to achieve desired monomer and enzyme concentrations. Fullyfunctionalized electrodes were rinsed in PBS and air-dried prior tostorage at 4° C.

Example 7: Dual-Electrode Functionalization

All stereoelectrode functionalization steps were performed by immersingboth electrode tips in functionalization solutions simultaneously. Thefirst two functionalization steps (i.e pretreatment and Pt Black)deposition were implemented on both microelectrode tips in sequenceusing the three electrode setup and functionalization solutionsdescribed previously. Enzyme biofunctionalization and permselectivelayer deposition were achieved using EDOT and o-AP solutions doped withglucose oxidase and lactate oxidase for electrodes 1 and 2 respectively.A total of four consecutive steps were used; both enzymaticbio-functionalizations were completed first, followed by growth ofpermselective layers. Appropriately doped PEDOT and PoAP layers werespatially confined to their respective electrodes via potential cyclingat the electrode of interest as described earlier. In each case, theopposing electrode was held at a constant potential of 0 V to preventnon-specific adsorption of the enzymatic transducer or oxidized monomeronto the tip. As an added precaution, the entire electrode shank wasrinsed in ultrapure water after each deposition step and in PBS aftercompletion of all four functionalization steps.

Example 8: Electrochemical Measurements

Amperometric sensitivity of both micro and macro electrode based sensorswas determined by measuring H₂O₂ oxidation current at a constant workingpotential of 0.5V upon sequential additions of requisite analyte stocksolution to a known volume of PBS. Following each addition, measuredcurrent signal was allowed to reach steady state for at least 10seconds. Plots of average current versus analyte concentration were usedto represent sensor response, and linear regression was used to estimatesensitivity, and linear sensing range (R2>0.98) Limit of detection foreach sensor was computed using the 3σ (i.e., 99% confidence) method.Sensor tunability was determined by measuring glucose response aftervarying number of PEDOT functionalization cycles using 1.6 mm diameterPt disk electrodes. Sensor sensitivity was normalized to account forvariations in available electroactive surface area after multiplefunctionalization and cleaning steps.

Example 9: Controlling Biomolecule Deposition in Space

The above described methods allow for controlling biomolecule depositionin space and are able to tune a level of molecule-doping based on numberof cycles of polymer growth. That enables biomaterial deposition on anyconductive surface irrespective of shape and organization, as well as 3Dpatterning of biomolecules. Once patterned, the biomolecules can be usedto detect analytes using electrical methods or other available detectionmodalities including but not limited to phosphorescence, fluorescence,SPR and Mass Spectrometry.

What is claimed is:
 1. A multi-analyte sensor, the sensor comprising: aplurality of sensing electrodes, each sensing electrode comprising atleast one electrically conductive material and being functionalized witha different enzyme composition from other sensing electrodes of theplurality, wherein at least two of the plurality of sensing electrodesare spaced apart at a microscale level prior to and afterfunctionalization and there is negligible cross-talk between theplurality of sensing electrodes, wherein each sensing electrodecomprises: a first layer comprising a first electrically conductivematerial, a second layer comprising a second electrically conductivematerial, and a third layer comprising a third electrically conductivematerial; wherein conductivity of the third electrically conductivematerial is pH dependent, and wherein the first layer is platinum black,the second electrically conductive biocompatible material ispoly(3,4-ethylenedioxythiophene), and the third electrically conductivematerial is poly(o-aminophenol).
 2. The multi-analyte sensor accordingto claim 1, wherein for each sensing electrode respectively, each of thedifferent enzymes is present in the second and third layers.
 3. Themulti-analyte sensor according to claim 1, wherein the multi-analytesensor further comprises a control unit that comprises first and secondelectrodes, wherein the first electrode is a reference electrode and thesecond electrode is a counter electrode.
 4. The multi-analyte sensoraccording to claim 1, wherein the multi-analyte sensor further comprisesa control unit that is a potentiostat.
 5. A sensor comprising: at leastone sensing electrode, wherein at least a portion of the at least onesensing electrode comprises: a first layer comprising a firstelectrically conductive material; a second layer comprising a secondelectrically conductive material and an enzyme; and a third layercomprising a third electrically conductive material and the enzyme;wherein conductivity of the second electrically conductive biocompatiblematerial is pH dependent; and wherein the first layer is platinum black,the second electrically conductive material ispoly(3,4-ethylenedioxythiophene), and the third electrically conductivematerial is poly(o-aminophenol).
 6. The sensor according to claim 5,wherein the sensor further comprises a control unit that is apotentiostat.
 7. The sensor according to claim 5, further comprising asecond sensing electrode comprising: a first layer comprising a firstelectrically conductive material; a second layer comprising a secondelectrically conductive material and a second enzyme; and a third layercomprising a third electrically conductive material and the secondenzyme, wherein conductivity of the third electrically conductivematerial is pH dependent.
 8. The sensor according to claim 7, whereinthe sensing electrodes are spaced apart at a microscale level.